Correction: In ovo inoculation of Bacillus subtilis and raffinose affects growth performance, cecal microbiota, volatile fatty acid, ileal morphology and gene expression, and sustainability of broiler chickens (Gallus gallus)

Abstract

Missing in-text citation "[Immediately following euthanasia, cecal content samples were collected and kept frozen until VFA analysis according to the procedures described by Saad et al.(33). Immediate freezing is a validated protocol in avian studies to stop microbial metabolic activity and prevent short-chain fatty acids from breaking down or evaporating during storage (34). The concentrations of VFA were measured using a mass spectrometer Agilent 5975C, carrier gas helium, column HP-5 ms (30m x 250µm x 0.25µm), and temperature: 35 o C/3 min, 5 o C/min to 250 o C for 3min, total 49 min, carrier gas helium 1ml/min constant speed; split ratio 30:1.]"2. [Chalalai T, Promsut W, Hinkhao K, Hengphrathani T, Sangsakul K, Bhavabhutanon N, et al. Effects of probiotics and amprolium on performance, lesion scores, oocyst shedding, and histopathological changes in Eimeria tenella-infected broiler chickens. Vet World. 2025;18(6):1400.] was not cited in the article. The citation has now been inserted in the section [DISCUSSION,Paragraph Number 3] and should read: "The antimicrobial effects of B. subtilis, raffinose, and their synbiotics are well-presented in the current study. The inhibitory activity of these supplements is attributed to their ability to produce antibacterial and antifungal substances, including bacteriocins, bacteriocin-like substances, acetic acid, hydrogen peroxide, carbon dioxide, diacetyl, and lactic acid (7,56). Intestinal immunity modulation (7), fat storage regulation (57), dietary fiber utilization (58), and competitive insularity of pathogenic bacteria (59) are other functional properties of the antimicrobial role of the in ovo supplements. The impacts of probiotics and probiotics on modifying the intestinal microbiota composition by suppressing pathogen numbers and elevating counts of beneficial microorganisms were previously documented ( 14). Oh et al. propose that a similar mode of action may explain the results given here, with the resulting improvement in broiler chicken health and performance. However, the administration of Amprolium (75 g/100 L) during the second week was implemented as a standard commercial prophylactic measure to manage coccidial risk (65). While anticoccidials can influence gut microbiota and immune responses, Amprolium was applied uniformly across all experimental groups, thereby balancing its physiological effects within the randomized design. Furthermore, current research suggests that B. subtilis and Amprolium are not mutually exclusive; B. subtilis does not rely on the thiamine-dependent pathways targeted by Amprolium in Eimeria parasites (65,66). Therefore, the use of Amprolium in this study reflects a realistic commercial environment and further validates the effectiveness of the synbiotic under standard production stressors, with no significant confounding interactions. However, we suggest that further investigation is required to improve combination therapies in clinical settings." and the production of SCFAs improves the competition with the pathogens. It improves the intestinal histomorphology and immune related genes. This has been demonstrated before; for instance, treatment with beneficial bacteria reduced the virulence of pathogenic bacteria by promoting the formation of SCFAs in the gut (64). We propose that a similar mode of action may explain the results given here, with the resulting improvement in broiler chicken health and performance. However, the administration of Amprolium (75 g/100 L) during the second week was implemented as a standard commercial prophylactic measure to manage coccidial risk (65). While anticoccidials can influence gut microbiota and immune responses, Amprolium was applied uniformly across all experimental groups, thereby balancing its physiological effects within the randomized design.Furthermore, current research suggests that B. subtilis and Amprolium are not mutually exclusive; B. subtilis does not rely on the thiamine-dependent pathways targeted by Amprolium in Eimeria parasites (65,66). Therefore, the use of Amprolium in this study reflects a realistic commercial environment and further validates the effectiveness of the synbiotic under standard production stressors, with no significant confounding interactions. However, we suggest that further investigation is required to improve combination therapies in clinical settings." and CD3 in the gut of broiler chickens while expressions of TLR-4, IL-1β, and IL-10 were not altered. The authors attributed this lack of improvement of innate immunity to the absence of pathogenic infections because the birds were reared in a sanitary environment. We selected b-actin as the reference gene for normalizing the expression of intestinal function (Mucin-2, VEGF), nutrient transporter (SGLT1, EAAT3), and immune (IL-2, TLR-4) genes due to its demonstrated stability in chicken ileal studies and its prevalent use among leading experts in the field (85,86). Nonetheless, we acknowledge that using a single housekeeping gene without rigorous validation may not adequately satisfy MIQE reporting guidelines. We have used the standard 2 -ΔΔCT method for relative quantification; however, in future investigations, we intend to incorporate additional reference genes to enhance the robustness and transparency of our gene expression results." revealed that in ovo injection of raffinose up-regulated the expression of chB6 and CD3 in the gut of broiler chickens while expressions of TLR-4, IL-1β, and IL-10 were not altered. The authors attributed this lack of improvement of innate immunity to the absence of pathogenic infections because the birds were reared in a sanitary environment. We selected b-actin as the reference gene for normalizing the expression of intestinal function (Mucin-2, VEGF), nutrient transporter (SGLT1, EAAT3), and immune (IL-2, TLR-4) genes due to its demonstrated stability in chicken ileal studies and its prevalent use among leading experts in the field (88,89). Nonetheless, we acknowledge that using a single housekeeping gene without rigorous validation may not adequately satisfy MIQE reporting guidelines. We have used the standard 2 -ΔΔCT method for relative quantification; however, in future investigations, we intend to incorporate additional reference genes to enhance the robustness and transparency of our gene expression results."There was a mistake in The original version of this article has been updated.There was a mistake in The original version of this article has been updated. A correction has been made to the section [MATERIALS AND METHODS, Bacteriological examination]. The incorrect paragraph is below:One gram of each cecal sample was homogenized in 9 ml of sterilized saline peptone solution and stirred for 30 min to obtain 10 -1 dilution. Decimal serial dilutions were prepared from the previous (10 -1 )to 10 -7 .According The corrected paragraph is below:"[One gram of each cecal sample was homogenized in 9 ml of sterilized saline peptone solution and stirred for 30 min to obtain 10 -1 dilution. Decimal serial dilutions were prepared from the previous (10 -1 ) to 10 after incubation at 30˚C for 48 h (30). Sabouraud Dextrose Agar (SDA) was used to enumerate total yeasts and molds count (TYMC) after incubation at 30˚C at 24 h for yeasts and 25˚C for 5 days for fungi (32).Violet red bile agar, MacConkey agar, and Bacillus cereus agar (Oxoid) were used for total coliforms, Escherichia coli, and B. subtilis, respectively, after incubation at 37˚C for 24 h. Bacillus cereus agar was used for counting B. subtilis after incubation for 24 h at 37°C. Bacillus cereus agar was used for its selective properties to inhibit the growth of other bacteria, and that B. subtilis colonies were further identified and confirmed based on their distinct morphology and biochemical tests. MRS medium and Chromocult enterococci agar were used for lactic acid bacteria and Enterococcus spp., respectively after incubation at 37°C for 48 h. The microbial counts were converted into log10 CFU g -1 .]"The original version of this article has been updated. The incorrect paragraph: "[One gram of each cecal sample was homogenized in 9 ml of sterilized saline peptone solution and stirred for 30 min to obtain 10 -1 dilution. Decimal serial dilutions were prepared from the previous (10 -1 ) to 10 -7 . According to Abd and Alagawany et al.( 31), an aliquot of 0.1 mL of each dilution was spread over different specific media such as Plate count agar (PCA) for total bacterial count (TBC) after incubation at 30˚C for 48 h (30). Sabouraud Dextrose Agar (SDA) was used to enumerate total yeasts and molds count (TYMC) after incubation at 30˚C at 24 h for yeasts and 25˚C for 5 days for fungi (32). Violet red bile agar, MacConkey agar, and Bacillus cereus agar (Oxoid) were used for total coliforms, Escherichia coli, and B. subtilis, respectively, after incubation at 37˚C for 24 h. Bacillus cereus agar was used for counting B. subtilis after incubation for 24 h at 37°C.MRS medium and Chromocult enterococci agar were used for lactic acid bacteria and Enterococcus spp., respectively after incubation at 37°C for 48 h. The microbial counts were converted into log10 CFU g -1 .]"The corrected paragraph: "[One gram of each cecal sample was homogenized in 9 ml of sterilized saline peptone solution and stirred for 30 min to obtain 10 -1 dilution. Decimal serial dilutions were prepared from the previous (10 -1 ) to 10 -7 . According to Abd and Alagawany et al.( 31), an aliquot of 0.1 mL of each dilution was spread over different specific media such as Plate count agar (PCA) for total bacterial count (TBC) after incubation at 30˚C for 48 h (30). Sabouraud Dextrose Agar (SDA) was used to enumerate total yeasts and molds count (TYMC) after incubation at 30˚C at 24 h for yeasts and 25˚C for 5 days for fungi (32). Violet red bile agar, MacConkey agar, and Bacillus cereus agar (Oxoid) were used for total coliforms, Escherichia coli, and B. subtilis, respectively, after incubation at 37˚C for 24 h. Bacillus cereus agar was used for counting B. subtilis after incubation for 24 h at 37°C.Bacillus cereus agar was used for its selective properties to inhibit the growth of other bacteria, and that B. subtilis colonies were further identified and confirmed based on their distinct morphology and biochemical tests. MRS medium and Chromocult enterococci agar were used for lactic acid bacteria and Enterococcus spp., respectively after incubation at 37°C for 48 h. The microbial counts were converted into log10 CFU g -1 .]"The original version of this article has been updated. The original version of this article has been updated.A correction has been made to the section [MATERIALS AND METHODS, Quantitative Real-TimeThe incorrect paragraph: "[Total RNA was extracted from the ileum using Trizol (Invitrogen;Thermo Fisher Scientific, Inc.) and then reverse-transcribed to cDNA using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems™, Waltham, MA, United States)following the manufacturer's protocol. Real-time RT-PCR was performed in an Mx3005P Real-Time PCR System (Agilent Stratagene, United States) using TOPreal™ qPCR 2 × PreMIX (SYBR Green with low ROX) (Enzynomics, Korea) following the manufacturer's instructions and according to the previous studies (34-36). The PCR cycling conditions included an initial denaturation at 95°C for 12 min followed by 40 cycles of denaturation at 95°C for 20 s, annealing at 60°C for 30 s, and with an extension at 72°C for 30 s. The expression level of the target genes was normalized using the mRNA expression of a known housekeeping gene, Bactin. Results are expressed as fold changes compared to the control groups following the 2 - ΔΔCT method (37). The primer sequences used are given in Table 2.]"The corrected paragraph: "[Total RNA was extracted from the ileum using Trizol (Invitrogen; Thermo Fisher Scientific, Inc.) and then reverse-transcribed to cDNA using a High-Capacity cDNA ReverseTranscription Kit (Applied Biosystems™, USA) following the manufacturer's protocol. Real-time RT-PCR was performed in an Mx3005P Real-Time PCR System (Agilent Stratagene, USA) using TOPreal™ qPCR 2X PreMIX (SYBR Green with low ROX) (Enzynomics, Korea) following the manufacturer's instructions and according(35-37). The PCR cycling conditions included an initial denaturation at 95°C for 12 minutes followed by 40 cycles of denaturation at 95°C for 20 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for 30 seconds. All qPCR tests were validated using melting curve analysis, which confirmed a single, distinct peak for each target gene, including b-actin, thereby ensuring high primer specificity and the absence of nonspecific amplification or primer dimers. To ensure transparency in gene expression analysis, mRNA levels were normalized to b-actin, and fold changes were calculated using the 2 -ΔΔCT (38). The primer sequences used are given in Table 2.]"The original version of this article has been updated. per treatment to represent all treatment replicates) to ensure biological diversity. Because only one bird was sampled per cage (replicate), there was no nesting of multiple birds within a single replicate for these specific measurements, which simplifies the variance structure. We acknowledge that the absence of a mixed model for nesting effects represents a simplified variance structure; however, birds were randomly selected from separate replicates to maintain biological independence and minimize potential unstable inference. This approach ensures that the degrees of freedom are correctly partitioned and that Type I error is not inflated by treating individual birds within a cage as independent observations due to the use of Tukey (HSD) for means comparison. Data are presented as means ± SEM, and the significance was declared at P<0.05.]"The original version of this article has been updated. We propose that a similar mode of action may explain the results given here, with the resulting improvement in broiler chicken health and performance. However, the administration of Amprolium (75 g/100 L) during the second week was implemented as a standard commercial prophylactic measure to manage coccidial risk (65). While anticoccidials can influence gut microbiota and immune responses, Amprolium was applied uniformly across all experimental groups, thereby balancing its physiological effects within the randomized design.Furthermore, current research suggests that B. subtilis and Amprolium are not mutually exclusive; B.subtilis does not rely on the thiamine-dependent pathways targeted by Amprolium in Eimeria parasites (65,66). Therefore, the use of Amprolium in this study reflects a realistic commercial environment and further validates the effectiveness of the synbiotic under standard production stressors, with no significant confounding interactions. However, we suggest that further investigation is required to improve combination therapies in clinical settings.]"The original version of this article has been updated.A correction has been made to the section [DISCUSSION, Paragraph Number 8]:The incorrect paragraph: [Early establishment of beneficial microbiota and modulation of the immune system in poultry by in ovo administration of probiotics, prebiotics, or synbiotics contribute to increasing general health, well-being, and performance of birds. These supplements also contribute to eliminating the use of prophylactic drugs. The immunomodulating capabilities of these supplements are represented in several reports (9,75,81). TLR-2 is a pathogen recognition receptor expressed on infectious agents to recognize the molecular patterns associated with microbes and play a vital role in initiating and regulating the innate immune response. IL-2 plays a fundamental role in stimulating the proliferation of T and B lymphocytes and natural killer cells as a pro-inflammatory cytokine. In this study, we evaluated the expression of TLR-2 and IL-2 as indicators of the effect of in ovo supplements on innate and adaptive immune responses, respectively. Both TLR-4 and IL-2 were downregulated in the present study by in ovodelivered supplements. The downregulation of the expression of these genes could be a result of the inhibitory impacts of in ovo supplements to pathogen colonization, which eliminates the need for TLR-4